The present invention relates to an agent which is utilized for immunological assays in medical fields for blocking nonspecific adsorption, a process for preparing thereof and a method of blocking nonspecific adsorption.
In recent years, immunological techniques such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blot technique and the like are often utilized in the fields of clinical tests, immunology, biochemistry, molecular biology and the like.
In these techniques, the desired material is fixed and assayed by fixing the object material to a solid phase such as a plate, a nitrocellulose membrane, etc., by adding an isotope or an enzyme-labelled antibody (a probe) which binds specifically to the material and then by determining its radiation level or enzyme activity. Then, it is important that the probe reacts with the object material alone and the probe can not adsorb specifically to the solid phase.
Therefore, after fixing the object material to the solid phase, the absorbing point on the solid phase must be closed by adding a blocking agent (an agent for blocking nonspecific adsorption). As an example, in the case of ELISA, the blocking agent is added after antigen is fixed to the plate, and then the probe is added. The blocking agent serves to prevent nonspecific adsorption of the probe to the plate.
Usually, a solution of bovine serum albumin (BSA) is used as the blocking agent. It needs to dissolve BSA in concentration more than 1% generally, and in concentration more than 2%, preferably about 5% for perfectly blocking nonspecific adsorption. BSA is a protein which is easily available in relatively large amount. However, BSA contributes to the high cost of analysis because it takes long time for dissolving BSA in the solution and because its price is high.
However, any material, which has no mutual interaction against the object material or a probe and can adsorb well to a solid phase, can act as a blocking agent theoretically. Accordingly, it is desired to develop an inexpensive and excellent blocking agent.
In 1984, Johnson et al reported that nonfat dry milk (SMP) was superior to BSA for blocking nonspecific adsorption in Southern blot analyses, and a 5% SMP solution acted as an efficient blocking agent in ELISA (Gene Anal. Techn. Vol. 1. pp. 3-8, 1984). Further, Miskimins et al reported that a 5% SMP solution could be an efficient blocking agent (Proc. Natl. Acad. Sci. U.S.A. Vol. 82. pp. 6741-6744, October 1985). Then, Ohta described the efficiency of SMP in the introduction of cloning technology in which expressed vector .lambda.gt11 was used (Saibo Kogaku, Vol. 5, No. 3, pp264-270, 1986).
In the above publications, SMP is used as a blocking agent which is dissolved in Tris buffer solution. If the solution of SMP is used as it is, its merchandise value for a reagent is remarkably lowered by the milky turbidity of the solution. When the blocking agent is mass-produced and is on the market as merchandise, it is more advantageous under normal temperature conditions than in low temperature conditions in the cost-effectiveness. In the normal temperature conditions, the SMP solution should be sterilized by an autoclave and the like. However, when SMP was merely dissolved in water or a buffer solution of Tris or phosphate, it was found that its merchandise value was lost because the solution was browned by heating, the turbidity of the solution was increased and a precipitate was ocationally produced from the solution.